The present invention generally relates to the field of oncology and inhibitors of Bcl-2 proteins, and more particularly to small molecule inhibitors of Bcl-2 proteins involved in mediating the death of cancer cells, virally infected cells and self-reactive lymphocytes.
Bcl-2 (B cell lymphoma/leukemia 2) was originally identified at the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas. Bcl-2 is now known to belong to a growing family of proteins which regulate programmed cell death or apoptosis. The Bcl-2 family includes both death antagonists (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, Brag-l, Mcl-l and Al) and death agonists (Bax, Bak, Bcl-x5, Bad, Bid, Bik and Hrk) (Thompson, Science 267:1456-62 (1992); Reed, J. Cell Biol. 124:1-6 (1994); Yang et al., Blood 88:386-401 (1996)). This family of molecules shares four homologous regions termed Bcl homology (BH) domains BH 1, BH2, BH3, and BH4. All death antagonist members contain the BH4 domain while the agonist members lack BH4. It is known that the BH1 and BH2 domains of the death antagonists such as Bcl-2 are required for these proteins to heterodimerize with death agonists, such as Bax, and to repress cell death. On the other hand, the BH3 domain of death agonists is required for these proteins to heterodimerize with Bcl-2 and to promote apoptosis.
Programmed cell death or apoptosis plays a fundamental role in the development and maintenance of cellular homeostasis. Homologous proteins and pathways in apoptosis are found in a wide range of species, indicating that cellular demise is critical for the life and death cycle of the cell in all organisms. When extracellular stimuli switch on the cell-death signal, the response of the cell to such stimuli is specific for the particular cell type (Bonini et al., Cell 72:379-95 (1993)). The pathway to cellular suicide is controlled by certain checkpoints (Oltvai, Cell 79:189-92 (1994)). The Bcl family proteins, including both antagonists of apoptosis (such as Bcl-2) and agonists of apoptosis (such as Bax), constitute the primary checkpoint. As such, the transmission of a cell-death signal can be either promoted or blocked by the different combinations of the Bcl-2 family members. The three-dimensional structure of a death antagonist, Bcl-XL, as determined by X-ray crystallography and NMR spectroscopy, provides a structural basis for understanding the biological functions of Bcl-2 family members and for developing novel therapeutics targeting Bcl-2 mediated apoptotic pathways (Muchmore et al., Nature 381:335-41 (1996)).
The detailed mechanism of Bcl-2 proteins in mediating molecular pathways of apoptosis has been the subject of intensive investigation. It is known that the apoptotic signaling pathway involves the activation of caspases which, once activated, cleave several cellular substrates such as poly(adenosine diphosphate-ribose) polymerase (PARP) and lead to final events of apoptosis. Bcl-2 plays a crucial role in regulating the process of apoptosis. One possible mechanism for Bcl-2 function is that Bcl-2 inhibits the release of cytochrome c from mitochondria. Cytochrome c is important for the activation of caspases. As such, Bcl-2 blocks caspase activation and subsequent events leading to apoptosis.
Being able to block apoptosis, Bcl-2 is known to contribute to neoplastic cell expansion by preventing normal cell turnover caused by physiological cell death mechanisms. High levels and aberrant patterns of Bcl-2 gene expression are found in a wide variety of human cancers, including xcx9c30-60% of prostate, xcx9c90% of colorectal, xcx9c60% of gastric, xcx9c20% of non-small cell lung cancers, xcx9c30% of neuroblastomas, and variable percentages of melanomas, renal cell, and thyroid cancers, as well as acute and chronic lymphocytic and non-lymphocytic leukemias (Ellis et al., Cell Biol. 7, 663 (1991); Henkart, Immunity 1, 343 (1994)); Kxc3xa4gi et al., Science 265, 528 (1994); Kxc3xa4gi et al., Nature 369, 31 (1994); Heusel et al., Cell 76, 977 (1994)).
The expression levels of Bcl-2 protein also correlate with relative resistance to a wide spectrum of current chemotherapeutic drugs and xcex3-irradiation (Hanada et al., Cancer Res. 53:4978-86 (1993); Kitada et al., Antisense Res. Dev. 4:71-9 (1994); Miyashita et al., Cancer Res. 52:5407-11 (1992); Miyashita et al., Blood 81:151-7 (1993)). Since Bcl-2 can protect against such a wide variety of drugs which have very different mechanisms of action, it is possible that all these drugs use a common final pathway for the eventual induction of cell death which is regulated by Bcl-2. This notion is supported by the findings that chemotherapeutic drugs induce cell death through a mechanism consistent with apoptosis as opposed to necrosis. Therefore, Bcl-2 can inhibit the cell killing effect of currently available anticancer drugs by blocking the apoptotic pathway.
Because of its role in blocking apoptosis, Bcl-2 plays an important role in many types of cancer. As noted above, Bcl-2 blocks apoptosis, thereby preventing normal cell turnover. As a result, neoplastic cell expansion occurs unimpeded by the normal cellular turnover process. Prostate cancer is one particular example where Bcl-2 has important implication in the pathogenesis and treatment for a disease. Approximately 100,000 new cases of prostate cancer are diagnosed each year in the United States and about 30,000 deaths per year are attributable to this disease (Lynn et al., JNCI 87:867 (1995)). It has recently been found that hormone therapy-resistant prostate cancers express Bcl-2 (McDonnell et al., Cancer Res. 52:694-04 (1992)), while the normal prostate cells from which prostate cancers originate lack Bcl-2 (Colombel et al., Am J Pathol 143:390-400 (1993)). This indicates that Bcl-2 may protect prostate cancer cells from undergoing apoptosis induced by the anticancer drugs, such as Taxol (Haldar et al., Cancer Res., 56:1235-5 (1996)). The clinical efficacy of nearly every cytotoxic anticancer drug currently available depends directly or indirectly on the assumption that tumor cells grow more rapidly than normal cells. However, this may not apply to human prostate cancer cells, which show very slow growth kinetics. Tumor kinetics studies have indicated that prostate cancer may be the consequence of the imbalance in cell turnover mechanisms more so than an increase in cell cycle rates. Thus, current anticancer drugs may not be effective in eradicating these nonproliferative prostate cancer cells.
The understanding of the biology of Bcl-2 in cancer and chemoresistance has opened new avenues in the development of novel anticancer strategies. One effective approach to overcome the chemoresistance of prostate cancers is to inhibit the protective function of Bcl-2 proteins. New drugs that modulate Bcl-2 mediated apoptotic response would represent a novel mechanism-based strategy for the treatment of prostate cancers and other cancers. Because the function of Bcl-2 is not absolutely necessary in many normal cell types (Veis et al., Cell, 75:229-40(1993)), a systematic inhibition of Bcl-2 may not affect the normal cellular function. This notion is supported by recent encouraging data from the clinical trial that antisense oligonucleotides targeted against the Bcl-2 gene can specifically inhibit non-Hodgkin""s lymphoma in humans (Webb et al,. Lancet 349:1137-41 (1997)). However, the clinical value of such antisense oligonucleotides is limited by their lack of enzymatic stability, cell permeability, and oral activity. As discussed above, currently available anticancer drugs may not be effective due to the chemoresistance of prostate cancer cells. Therefore, there is an impending need for highly potent, cell permeable, and orally active Bcl-2 inhibitors as a new generation of effective therapeutics for the treatment of prostate cancer, as well as other cancers.
Compared to other therapeutics such as antibodies, peptides or antisense oligonucleotides, small organic drugs may possess several advantages in the clinical application: (1) they are less likely to be immunogenic; (2) they are likely to be stable and to be able to cross the cell membrane; (3) they are more likely to be administrable through the oral route, which is most desirable in terms of patient compliance; and (4) they are amenable to synthesis and modification which significantly lowers the cost of the therapeutic treatment.
It is an object of the invention to provide small molecule inhibitors of bcl-2 function useful in treatment of cancer, autoimmune disease and certain types of viral infection which are characterized by cellular signals which inhibit apoptosis.
It is an object of the invention to induce apoptosis of cells, particularly cancer cells, most particularly cancer cells which are regulated by Bcl-2.
It is an object of the invention to provide novel therapeutics and methods of treatment for reversing Bcl-2-mediated blockage of cell apoptosis in cancer cells.
It is an object to provide of the invention to overcome Bcl-2-mediated chemoresistance in tumor cells.
These and other objects of the invention are apparent from the following description.
A method of inducing apoptosis of cells regulated by Bcl-2 in a subject is provided. An effective amount of an active compound is administered to the subject. Preferably, the compound causes the fragmentation of DNA in a Bcl-2 transfected HL-60 cell line when incubated with such cells at a concentration of not more than 100 xcexcM for 24 hours. In some embodiments, the compound is also characterized by a dissociation constant KD of not more than about 500 xcexcM, preferably no more than about 100 xcexcM, most preferably no more than about 10 xcexcM, for binding the hydrophobic pocket on the Bcl-2 protein formed by the BH1, BH2, and BH3 domains.
By xe2x80x9cregulated by Bcl-2xe2x80x9d with respect to the condition of a cell is meant that the balance between cell proliferation and apoptotic cell death is controlled, at least in part, by Bcl-2. By xe2x80x9capoptotic cell deathxe2x80x9d is meant the programed death which results in controlled autodigestion of the cell, as opposed to necrotic cell death. Apoptotic cell death is characterized by cytoskeletal disruption, cell shrinkage, and membrane blebbing. The nucleus undergoes condensation and nuclear DNA becomes degraded and fragmented. Apoptosis is also characterized by loss of mitochondrial function. Necrotic cell death, on the other hand, is a pathological form of cell death resulting from acute cellular injury, which is typified by rapid swelling and lysis.
According to certain embodiments of the invention, the cells induced to undergo apoptosis comprise cancer cells, virus-infected cells or self-reactive lymphocytes. Thus, the active compounds are used to treat cancer, viral infection, or autoimmune disorders.
In another embodiment, a method of reversing Bcl-2-mediated blockage of apoptosis in cancer cells is provided by contacting such cells with an active compound of the invention. In another embodiment, a method is provided for treating a subject afflicted with a cancer characterized by cancer cells which express Bcl-2. The method comprises administering an effect amount of an active compound of the invention.
Active compounds which have a molecular weight in the range of from about 150 to about 500 daltons.
According to one embodiment of the invention, the compounds have the formula I: 
wherein:
X is selected from the group consisting of CH2; CHOCH3; NH; O and S;
Y and Z are independently selected from the group consisting of CH and N; and when Z is N, then Y may further be xe2x80x94CR6, where R6 is selected from the group consisting of CH3; OCH3; CNH2; and COH;
R1 is selected from the group consisting of hydrogen; C1-5 alkyl; C1-5 alkoxy; OH; NH2; NO2; CHO; COCH3; COOH; COOCH3; N(C1-3 alkyl)2; NH(C1-3 alkyl); OCOCH3; OCOCH2CH3; NHCOCH3; NHNHCOCH3; NHNHCONH2; phenyl; phenyl which is mono-, di-, or tri-substituted with NH2, OH, halogen, NO2, CF3, COOH or COOCH3; cyclohexyl; cyclohexyl which is mono-, di-, ortri-substituted with NH2, OH, halogen or CF3; and five- and six-member heterocyclic rings, preferably a heterocyclic ring selected from the group consisting of piperidino, piperazino, morpholino, pyrimidyl, pyrrolidino and imidazo;
R2 is selected from the group consisting of hydrogen; C1-3 alkyl; C1-3 alkoxy; halogen; CF3; NH2; OH; COOH; COOCH3; CONH2; and CONHCH3;
or, R1 and R2 together may form the group xe2x80x94CH2CH2CH2xe2x80x94 or xe2x80x94CH2CH2CH2CH2xe2x80x94;
or, R1 and R2 together may form, starting from R1, the group xe2x80x94NHCH2CH2xe2x80x94, xe2x80x94NHCOCH2xe2x80x94, or xe2x80x94OCOCH2xe2x80x94;
R3 is selected from the group consisting of H; CH3; CF3; OCH3; NH2; OH; COOH; COCH3; CHxe2x95x90CH2; CH2xe2x95x90CHCH2; CH(CH3)2; CH2OH; CH2NH2; CH2COOH; cyclohexyl; cyclohexyl which is mono-, di-, or tri-substituted with NH2, OH, halogen, OCH3 or CF3; five- and six-member heterocyclic rings, preferably a heterocyclic ring selected from the group consisting of piperidinyl, piperazinyl, morpholino, pyrimidyl, pyrrolyl, pyrrolidino, and imidazyl; and a substituted phenyl group of the formula: 
xe2x80x83wherein
R7, R8 and R9 are independently selected from the group consisting of hydrogen, CH3, CF3, OH, OCH3, CH2OH and CHO; provided that at least two of the members of the group R7, R8 and R9 must be OH or OCH3 when the remaining member of the group is hydrogen, CH3 or CF3;
R4 and R5 are independently selected from the group consisting of hydrogen, CH3, and OCH3; and when Y and Z are both CH, R4 and R5 may be further selected from OH and NH2;
or, R4 and R5 together may form the group xe2x80x94CH2CH2CH2xe2x80x94 or xe2x80x94HH2CH2CH2CH2xe2x80x94;
or, R4 and R5 together may form, starting from R4, the group xe2x80x94NHCH2CH2xe2x80x94, xe2x80x94NHCOCH2xe2x80x94, xe2x80x94OCOCH2xe2x80x94 or xe2x80x94O(CH2)nxe2x80x94Oxe2x80x94, wherein n is 1, 2 or 3;
or a pharmaceutically acceptable salt thereof when the compound includes at least one NH2 or COOH substituent.
Preferably, R2 is CH3, CH2CH3, COOH, COOCH3, CONH2, or CONHCH3.
Preferably, R7, R8 and R9 are all OCH3; or R7 and R9 are OCH3, and R8is OH.
When R1 or R3 is substituted cyclohexyl, the preferred position of the substitution is para. Likewise, when R1 is substituted phenyl, the preferred position of the substitution is para.
The preferred group corresponding to R3 in formula I is the substituted phenyl group of the formula: 
Preferred compounds according to formula I include the compounds identified as HA11-1 through HA11-73, listed in Table 1, below. Most preferred compounds according to formula I include HA11-57 and HA11-17: 
According to another embodiment of the invention, the active compounds have the formula II: 
wherein
R1, R2, R3 and R4 are independently selected from the group consisting of hydrogen; C1-5 alkyl; C1-5 alkoxy; OH; NH2; NO2; CHO; COCH3; COOH; COOCH3; N(C1-3 alkyl)2; and NH(C1-3 alkyl); and one of R1, R2, R3 and R4 may be phenyl or a heterocyclic ring, preferably a heterocyclic ring selected from the group consisting of piperidino, piperazino, morpholino, pyrimidyl, pyrrolidino and imidazo; provided at least one of R1, R2, R3 and R4 must be hydrogen;
R5 and R6 are independently selected from the group consisting of hydrogen; CN; CH2CN; COOCH3; CONH2; phenyl; phenyl which is mono-, di-, or tri-substituted with NH2, OH, halogen, NO2, CH3, OCH3, CF3, COOH or COOCH3; cyclohexyl; cyclohexyl which is mono-, di-, or tri-substituted with NH2, OH, halogen or CF3; and five- and six-member heterocyclic rings, preferably a heterocyclic ring selected from the group consisting of pyrrolyl, imidazolyl, piperidinyl, piperazinyl, morpholino, pyrimidyl and pyrrolidino; provided, only one of R5 or R6 may be phenyl, substituted phenyl, cyclohexyl, substituted cyclohexyl or heterocyclic in the same compound, and further provided that when one of R5 or R6 is phenyl, substituted phenyl, cyclohexyl, substituted cyclohexyl or heterocyclic, then the other must be hydrogen;
or at least one of R5 and R6 may be halogen, provided that the other must be C1-5 alkyl or C1-5 alkoxy.
or a pharmaceutically acceptable salt thereof when the compound includes at least one NH2 or COOH substituent.
When R5 or R6 is substituted phenyl or substituted cyclohexyl, in formula II, the preferred position of the substitution is para.
Preferred compounds according to formula II include the compounds identified as HA12-3 and HA12-16 (compound HA12-16 may also be identified herein as xe2x80x9cHA01xe2x80x9d): 
In the compounds of formula I and II, where halogen substitution is possible, chorine, fluorine and bromine are preferred, with fluorine being most preferred.
According to another embodiment of the invention, the active compounds have the formula III: 
wherein:
X is selected from the group consisting of CH2; CHOCH3; NH; NCH3; O; and S;
R1 is selected from the group consisting of OH; NH2; CHO; COCH3; COOH; N(C1-3 alkyl)2; NH(C1-3 alkyl); OCOCH3; OCOCH2CH3; NHCOCH3; NHNHCOCH3; NHNHCONH2; N(C1-3alkyl)2; NH(C1-3 alkyl); and five- and six-member heterocyclic rings, preferably a heterocyclic ring selected from the group consisting piperidinyl, piperazinyl, morpholino, pyrimidyl, pyrrolyl, pyrrolidino and imidazyl;
R2 is selected from the group consisting of C1-3 alkyl; C1-3alkoxy; OH; NH2; CHO; COCH3; OCOCH3; OCOCH2CH3; COOH; COOCH3; COOCH2CH3; COOCH2CH2CH3;
R3 is selected from the group consisting of C1-3 alkyl; C1-3 alkoxy; CN; CH2CN; CH2NO2; CHO; COCH3; COOH; OCOCH3; OCOCH2CH3; NHCOCH3; NHNHCOCH3; NHNHCONH2; CHxe2x95x90CH2; CH2CHxe2x95x90CH2; CH2CHO; and five- and six-member heterocyclic rings, preferably a heterocyclic ring selected from the group consisting piperidinyl, piperazinyl, morpholino, pyrimidyl, pyrrolyl, pyrrolidino and imidazyl;
R4 is selected from the group consisting of C1-3 alkyl; C1-3 alkoxy; CN; CH2CN; CH2NO2; CHO; COCH3; COCH3; COOH; COOCH3;COOCH2CH3; COOCH2CH2CH3; OCOCH3; OCOCH2CH3;
R5 is selected from the group consisting of hydrogen CH3; OCH3; OH: NH2; Br; Cl; and F; and
R6, R7 and R8 are selected from the group consisting of hydrogen, CH3; CH2CH3; CF3; NH2; OH; OCH3; CN; NO2; Cl; Br; F; COOH; and COOCH3; provided, at least one member of the group R6, R7 or R8 must be Cl, Br or F when the remaining members of said group are hydrogen;
or a pharmaceutically acceptable salt thereof when the compound includes at least one NH2 or COOH substituent.
Preferred for formula III are the following:
R1: NH2; N(C1-3 alkyl)2; and NH(C1-3)alkyl; piperidinyl; piperazinyl; morpholino; pyrimidyl; pyrrolyl; pyrrolidino; and imidazyl;
R2: COCH3; OCOCH3; OCOCH2CH3; COOH; COOCH3; COOCH2CH3; and COOCH2CH2CH3;
R3: CN; CH2CN; CH2NO2; CHxe2x95x90CH2; CH2CHxe2x95x90CH2; and CH2CHO;
R4: COCH3; OCOCH3; OCOCH2CH3; COOH; COOCH3; COOCH2CH3; and COOCH2CH2CH3;
R5: hydrogen, Br; Cl; and F;
R6, R7 and R8: NH2; OH; OCH3; CN; NO2; Cl; Br and F.
When R6, R7 or R8 are Br, Cl or OCH3, the preferred positions of the substitution are R6 and R8.
According to another embodiment of the invention, the active compound for use in the method of the invention is selected from the group consisting of compounds HA13, HA14, HA02, HA03 and HA04: 